The oligosaccharide moiety of glycoconjugates play important roles in several biological processes of a cell, including the folding and transport of glycoproteins across cellular compartments. The oligosaccharide moieties bind to cellular proteins with high specificity and modulate the homo- and hetro-dimerization of glycoproteins. Due to the conformational flexibility of oligosaccharides, the torsional angles of a disaccharide unit, especially around the a1-6-linkage, adjust in such a way that the side groups of the oligosaccharides orient themselves in a manner that promotes favorable interactions with the binding residues of the protein. Branched oligosaccharides cross-link proteins and generate infinite networks of protein-carbohydrate complexes, resulting in the modulation of various cell responses. Defective glycan synthesis has been shown to have serious pathological consequences and result in several human diseases. Defining the oligosaccharide binding site of b-1,4-Galactosyltransferase(Gal-T1) by docking of oligosaccharides into the binding site: We have continued to use molecular modeling methods to study the binding of oligosaccharides to proteins, in particular the binding of various oligosaccharide substrates to Gal-T1, the 3D-structure of which has recently been determined in our laboratory, either in complex with UDP-galactose and manganese metal ion, or in complex with a-lactalbumin and N-acetylglucosamine (see Project # Z01 BC 009304-05 LECB ). A limited number of oligosaccharides have been shown to be the preferred substrates for Gal-T1. Among the different GlcNAc containing disaccharides only a b-linked disaccharide such as GlcNAcb1,4-GlcNAc or GlcNAcb1,2-Man are preferred over a-linked disaccharides. In fact a-methyl-GlcNAc is less preferred compared to GlcNAc by itself. Also, oligosaccharides such as N-glycans are more preferred acceptor substrates than a (GlcNAc)4. Examination of the GlcNAc binding site in Gal-T1 from the Gal-T1-LA-GlcNAc crystal structure reveals an "open canal shaped" extended sugar binding site that lies behind the GlcNAc binding site, with an average width and length of 10 A and 16 A, respectively. This site is formed by the residues from three regions; residues 280 to 289, residues 319 to 325 and residues 359 to 368. LA binds to this region in the crystal structure of Gal-T1-LA complex, therefore it is expected to compete with the GlcNAc containing oligosaccharides such as chitobiose. In order to understand the size and nature of the oligosaccharide binding site in Gal-T1 we have carried out a modeling study using various disaccharides and N-glycans to dock into the binding site of Gal-T1 without energy minimization of either of the molecules during docking. These studies indicate that GlcNAc with an a-linked substitution such as a-benzyl-GlcNAc can not binding to Gal-T1 since its binding creates a severe steric contacts with the highly conserved residue Tyr286, while as GlcNAc with a -linked substitution such as b-benzyl-GlcNAc can bind without any steric contacts. Docking of a biantennary N-glycan with GlcNAcs at its reducing ends in the extended sugar binding site reveals that the acceptor binding site in Gal-T1 can accommodate a linear pentasaccharide all the way from the GlcNAc moiety to the aspargine linked GlcNAc. The binding site can also accommodate either the a-1-3 arm (GlcNAcb1-2Mana1-3Man b1-4GlcNAcb1-4GlcNAc-N) or a-1-6 arm (GlcNAcb1-2Mana1-6Manb1-4GlcNAcb 1-4GlcNAc-N) of the N-glycan without any steric hindrance. In humans Gal-T1 family members are responsible for the synthesis of Gal moiety in different oligosaccharides, indicating that these enzymes, although transfer Gal to GlcNAc, recognize the remaining oligosaccharide moieties to which GlcNAc is attached to. Therefore the oligosaccharide binding site defined on bovine Gal-T1 will be important in understanding the structure-function of human Gal-T family members. The sequence comparison of the GlcNAc binding site of the family members reveals a little or no sequence variation, while the extended oligosaccharide binding region shows significant variations, indicating that these enzymes may prefer different GlcNAc containing oligosaccharides as their preferred sugar acceptors. However, the exact nature of their preferences based on the homology modeling and their crystal structure determination is underway. 1) Qasba PK. Carbohydrate Polymers 41, 293-309, 2000. Z01 BC 10041-04